This application addresses the Challenge area of Clinical research (04);Topic: A1-101: Develop novel methods and address key questions in mucosal immunology. The study of mucosal immunology of the anus (as with all mucosal epithelium) has been extremely challenging for numerous reasons: 1) the anus has a complex microbiologic environment which requires a level of immune tolerance to commensal organisms, 2) there are often frequent exposures to simultaneous pathogens as well as trauma induced by anal intercourse, 3) there has been a lack of techniques suitable to cohort studies to study immune parameters and 4) accessing tissue and fluids in longitudinal cohorts is often difficult. Anal mucosa has long been known to be vulnerable to sexually transmitted pathogens including HIV and human papillomavirus (HPV). Certainly, a greater understanding of these responses will be critical in the development of topical microbicides for sexually transmitted infection (STI) prevention as well as therapeutic interventions. In particular, therapeutic interventions are needed for viral pathogens such as HPV which is the most common STI and is responsible for 80-100% of anal cancers. Although HPV infections of the anus are common, even among women, most infection usually clear. Unfortunately, the risk of anal cancer continues for those with HPV persistence--the key in the development of all anogenital cancers. Fairly strong evidence suggests that immune mechanisms influence HPV clearance. Understanding the immunologic mechanisms that result in HPV persistence is critical in the development of therapeutic treatment for HPV-induced pre-cancers. In addition, HPV itself has been known to increase risks for HIV. Hence studying HPV may also give insight into mechanisms into which STIs contribute to HIV transmission. Over the past 15 years, we have attempted to address many of the immunologic questions in the cervix and vagina by incorporating immune assays in our ongoing longitudinal cohort: the natural history of HPV in teens (R37 CA51323) which began in 1990. This cohort undergoes close characterization for commensal and pathogenic organisms of the cervix, as well as behavioral interviews, at 4-month intervals. Over the years, we have adapted several assays focusing on innate and adaptive immunity that are suitable for study in the cohort including detection of cytokines and chemokines as well as measuring mRNA expression of Toll- like receptors (TLR) from cervical samples. In this well-established cohort, we have stored over 15,000 anal samples which have been collected since 1993 at 4 month intervals. We have the unprecedented and unique opportunity to examine mucosal immune responses over time in a well-characterized cohort. Aims of this study are to: 1) examine, in anal samples, the cytokines which are enhanced at time of HPV acquisition;2) examine in anal samples, the association between cytokines that mediate innate immunity (IFN-a, TNF, IL-6 and IL-8) and adaptive immunity (IFN gamma and IL 12) and clearance of incident HPV infection. Co-factors or behaviors that may also mediate cytokine and chemokine profiles will be examined including frequent anal intercourse, smoking cigarettes and oral contraceptive use and 3) compare cytokines and chemokine profiles between the anus and the cervix in response to HPV acquisition and clearance, and 4) to compare the cytokine/chemokine profiles between HPV infected women with normal and abnormal anal cytology. We have over 15,000 stored anal samples available for HPV DNA testing and cytokine/chemokine analysis for aims 1-3. All stored anal samples will be tested for HPV DNA. Cytokine and chemokine analysis will be performed from samples obtained from visits with an incident HPV infection. For aim 3, samples from the cervix of women for HPV DNA and cytokine/chemokine analysis are already available through the parent study for comparison. We also propose a subaim in which we will examine TLR expression from anal samples in women who acquire anal HPV. Since the current anal samples are not suitable for measuring TLRs, we have now started collecting these samples prospectively. We expect that we will have 300 samples available for examination for this aim. In summary, we have the unique opportunity to examine anal mucosal immune responses in a well characterized cohort using stored samples which will allow us to keep to our strict timeline. This data will give us a greater understanding of environmental factors that affect mucosal immune profiles. PUBLIC HEALTH RELEVANCE: This application plans to examine anal mucosal immune responses to human papillomavirus, tobacco use, anal intercourse and hormonal contraceptives.